The present inventions relates to metal ion affinity interactions with target molecules and, more particularly, to improvements thereof whereby non-specific interactions with non-target molecules can be diminished.
Affinity binding interaction based on the used of a chelated metal have been widely used due to the propensity of certain metals to preferably bind to given functional groups on target molecules such as proteins, peptides, and like compounds. Immobilized metal-ion chromatography, for example, has been used with a variety of different metals. Phosphorylated target molecules have been purified using columns containing immobilized ferric ions. Polyhistidine tagged fusion proteins nave been purified using columns containing immobilized divalent nickel. Peroxidase and biotin probes containing chelated metals have also been used for polyhistidine tagged fusion protein detection and in connection with immunoassays.
It is recognized that in metal-ion chromatography, the chelating functionality used to immobilize the metal to construct the chelator-metal conjugate is important. With iron, iminodiacetic acid functionality has been used due to its tight binding characteristics with ferric ions. With nickel, as well with many other less used metal ions, the nitrilotriacetic acid tetradentate functionality has been used in both column and probe formats due to its tight binding characteristics with these metal ions.
A Problem often encountered in metal ion affinity interactions is that of nonspecific binding, whereby the constructed chelator-metal ion conjugate, typically immobilized on a support or bound to a detection moiety such as an enzyme, e.g., a peroxidase, binds to non-target molecules as well as target molecules. Binding to non-target molecules can result in decreased sensitivity, nigh background, and overall poor assay performance.